The Harry M. Zweig Memorial Fund for Equine Research

Role of IgE in Skin Hypersensitivities of the Horse

Dr. Bettina Wagner

The term ‘allergy' is commonly used to refer to several types of diseases that are caused by different forms of hypersensitivity reactions. Hypersensitivity reactions are immune responses that are directed against harmless antigens, and that cause damage to host tissues, rather than destruction of an invading microorganism. Depending on the immune mechanisms involved in the hypersensitivity reactions, they are clustered in four different types (type I to type IV hypersensitivity). Type I hypersensitivities are mediated by a particular class of antibody known as Immunoglobulin E, or IgE. In contrast to other types of hypersensitivities, type I reactions are very quick to develop (e.g. bee sting or drug hypersensitivities, or certain food allergies, rhinitis and asthma). The target organs for type I hypersensitivities are often the gut, the lung, or the skin.

Immunoglobulin E has a natural protective function as a defense against parasites. However, IgE can also react in some individuals with harmless environmental antigens, called allergens. Some mammals, including humans, horses, dogs and cats seem to have a predisposition to develop IgE mediated allergies, while others, like cattle, sheep, pigs and rodents, are less affected.

In humans and in mouse models of induced IgE mediated allergies, type I hypersensitivities have been investigated extensively. In other species, including the horse, the mechanisms involved in type I hypersensitivities are less investigated. Although it is very likely that the general immune mechanisms leading to type I hypersensitivity are conserved between mammalian species, a more detailed analysis of the immune response of the horse is necessary to understand the specific aspects of equine allergic diseases and to improve the diagnosis and treatment of type I hypersensitivities in horses. In this project, we seek to provide the first direct evidence that IgE mediates type I allergies of the horse.

Equine IgE is believed to be involved in parasitic infections (Suter & Fey 1981) and in type I mediated allergic diseases of the horse (Matthews et al. 1983). IgE mediated mechanisms seem to play a role in many equine diseases like chronic obstructive pulmonary disease (McGorum et al. 1993, Schmallenbach et al. 1998), summer eczema (Larsen et al. 1988, Ungar-Waron et al. 1990), and urticaria or head-shaking (Mansfield et al. 1998). Many of these studies used intradermal injections of allergens to measure the hypersensitivity response in the horse

In humans, intradermal skin testing is a very powerful method to diagnose allergen specific type I hypersensitivity reactions. The injection of allergen induces the immediate release of preformed inflammatory mediators from mast cells in the skin. The reaction develops within minutes and is characterized by an increased wheal at the injection site and by a distinct flare of the surrounding area. Because of the nature of the equine skin and in particular its pigmentation, intradermal tests in the horse are more difficult to interpret due to the lack of flare. Other hypersensitivities, and in particular IgG mediated type II reactions may also induce similar reactions in the equine skin, and need to be distinguished from IgE mediated mechanisms to develop immunological based therapies to horse allergies.

Other methods to detect type I hypersensitivity responses are allergen specific tests that measure IgE in the blood sera of patients. While specific reagents to detect IgE are readily available for humans and rodents, determination of equine IgE was hampered for a long time by the lack of detection antibodies and pure reference proteins. To overcome the lack of reagents for the analysis of IgE responses in the horse, we have previously produced recombinant equine IgE. This recombinant IgE has a high structural and functional similarity to native IgE of the horse (Wagner et al. 2002a). We used the recombinant protein to immunize mice and generated monoclonal antibodies to horse IgE. These antibodies are highly specific reagents for the detection of horse IgE. They have been validated by several methods, including a sensitive ELISA for measuring IgE in equine serum, flow cytometry and microscopy for detection of IgE + cells in equine blood, and for affinity purification of native IgE from horse serum (Wagner et al. 2003).

In this project, we would take advantage of the anti-equine IgE antibodies to investigate IgE immune responses in horses with type I hypersensitivities and healthy control animals. The aims of the project are (1) to find the first direct evidence for the involvement of IgE in skin allergies, and (2) to develop an ELISA test to detect allergen specific IgE in the blood of horses with type I hypersensitivities.

Aim (1): To provide evidence that hypersensitivity is mediated by IgE in the horse, we would induce the reaction in non-allergic animals by transfer of IgE from an allergic horse. Serum IgE from a horse of the Baker Institute herd that developed a recurrent skin hypersensitivity during past summers, and from a healthy control animal, will be separated from IgG isotypes by an affinity purification method we established previously (Wagner et al. 2003). Skin testing will be performed to identify the allergen which is involved in the allergic reaction of the horse with the recurrent skin hypersensitivity. The purified IgE and IgG isotypes will be injected into the skin of healthy animals and allergen application will be used to provoke the hypersensitivity reaction in these non-allergic horses. Direct evidence for IgE mediated mechanisms will allow advances in treatments of equine hypersensitivities that target the causal IgE, instead of general immune suppression.

Aim (2): To establish an ELISA for allergen specific IgE detection in horse serum, blood samples will be collected from horses with skin hypersensitivities and urticaria. This part of the project will be performed in collaboration with Dr. William H. Miller ( Cornell University Hospital for Animals, CUHA) using blood samples of horses which are sent to the CUHA for intradermal skin testing. The skin testing provides information about the allergen(s) involved in the allergic response of the patients. The serum will be tested at the Baker Institute for allergen-specific IgE by ELISA to identify the antibodies which mediate the hypersensitivity response. We propose that the comparison of intradermal skin testing and allergen specific ELISA will allow a precise definition of cases induced by IgE mediated mechanisms, and also the exclusion of phenomena with similar clinical signs like IgG mediated type II hypersensitivities or cell mediated type IV hypersensitivities. The aim is to establish an ELISA that can be used for diagnosis of equine type I hypersensitivities.

Conclusion:

We expect to demonstrate for the first time the role of Immunoglobulin E in type I hypersensitivities (allergies) in the horse. This project should also provide the foundation for advanced methods of detection of equine allergies in clinical situations. Based on tests that distinguish between IgE mediated allergies and other types of hypersensitivity reactions, preventive applications to horse allergies may be developed in the future, e.g. vaccination strategies for horses with genetic predisposition to allergic reactions.